CN112430664A - Primer group, kit and method for detecting PML-RAR alpha fusion gene - Google Patents
Primer group, kit and method for detecting PML-RAR alpha fusion gene Download PDFInfo
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Abstract
The invention provides a primer group, a kit and a method for detecting PML-RAR alpha fusion gene, wherein the primer group comprises: a primer pair for PML-RARA fusion gene mutation and a corresponding fluorescent probe. When the primer group is used for carrying out fluorescent quantitative PCR detection, the mutation condition of the PML-RARA fusion gene can be detected through one-time PCR reaction, so that the detection difficulty of the mutation condition of the PML-RARA fusion gene is reduced.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer group, a kit and a method for detecting PML-RAR alpha fusion gene.
Background
Acute Promyelocytic Leukemia (APL) is a subtype of Acute Myelocytic Leukemia (AML) and accounts for about 7% -27% of AML. A large number of abnormal promyelocytes are present in the bone marrow of the patient, of which more than 95% are positive with specific chromosomal ectopic t (15; 17) (q 22; q21)/PML-RAR alpha fusion gene, i.e., PML gene on chromosome 15 and RARa gene on chromosome 17 form PML-RARa fusion gene. Translocation interactively rearranged the PML gene on 15q22 with the retinoic acid receptor alpha gene on 17q21 to form a PML-RARa fusion gene and an RARa-PML fusion gene on chromosome 15, respectively. According to the position of PML breakpoint, PML-RARa fusion gene has three subtypes bcr1, bcr2 and bcr3, which are respectively called long type (L type), variant type (V type) and short type (S type), and the probability of occurrence is 55%, 5% and 40% in sequence. Thus, the PML-RARA fusion gene is a typical molecular feature and a key causative factor of APL.
At present, methods for detecting the mutation of PML-RAR alpha fusion gene include probe hybridization, gene chip method and gene sequencing method.
However, the false positive rate of the probe hybridization method is high; the gene chip method has high test cost and complicated preparation method; the gene sequencing method has the disadvantages of higher test cost, more complicated operation and longer time consumption. Thereby increasing the difficulty of detecting the mutation condition of the PML-RARA fusion gene.
Disclosure of Invention
The invention provides a primer group, a kit and a method for detecting PML-RARA fusion gene, which can reduce the detection difficulty, cost and detection time of PML-RARA fusion gene mutation.
The invention provides a primer group for detecting PML-RAR alpha fusion gene, which comprises the following components:
the primer pair is used for amplifying the ABL reference gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.1, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.2, and the sequence of a corresponding fluorescent probe is shown as SEQ ID NO. 3;
a primer pair for amplifying a PML-RAR alpha L type (i.e., bcr1) fusion gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.4, the nucleotide sequence of a downstream primer is shown as SEQ ID No.7, and the sequence of a fluorescent probe is shown as SEQ ID No. 8;
a primer pair for amplifying PML-RAR alpha V type (i.e., bcr2) fusion genes, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.5, the nucleotide sequence of a downstream primer is shown as SEQ ID No.7, and the sequence of a fluorescent probe is shown as SEQ ID No. 8;
a primer pair for amplifying PML-RAR alpha S type (i.e., bcr3) fusion genes, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.6, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.7, and the sequence of a fluorescent probe is shown as SEQ ID NO. 8.
Whether the PML-RAR alpha fusion gene mutation and the PML-RAR alpha fusion gene genotype occur or not can be determined by the primer group consisting of the primer pair.
The real-time fluorescence quantitative polymerase chain reaction PCR has the characteristics of enhanced specificity, improved sensitivity, rapid detection, reduced pollution and the like. Based on this, the present invention also provides a kit for detecting a PML-RAR α fusion gene, comprising: the primer group, the synthetic reagent for synthesizing the first strand of cDNA, the fluorescent quantitative PCR premix for PCR test, the negative control sample and the positive control sample are adopted, wherein the negative control sample represents a sample without PML-RAR alpha fusion gene mutation, and the positive control sample represents a sample with PML-RAR alpha fusion gene mutation.
The invention adopts a real-time fluorescence quantitative PCR Taqman-MGB probe method to detect PML-RAR alpha fusion gene, thereby not only improving specificity, but also reducing cost; and the Taqman-MGB probe is superior to a common probe in the aspects of accuracy, repeatability, specificity, sensitivity and the like of an experimental result. Therefore, the detection method of the PML-RAR alpha fusion gene established by the invention has the advantages of accurate result, high specificity and sensitivity, no toxicity, no pollution, low cost, rapidness, high efficiency and the like.
In one embodiment of the present invention, the fluorescent probe represented by SEQ ID No.3 and the fluorescent probe represented by SEQ ID No.8 in the primer set are labeled with a fluorophore at the 5 'terminal region and a quencher at the 3' terminal region;
wherein the fluorescent group includes any one of FAM, TET, VIC, ROX, Texas Red-X, Cy3 and Cy 5.
The quencher group comprises: TAMRA, BHQ, DABCYL, and NFQ-MGB.
For example, the 5 ' end of the fluorescent probe shown in SEQ ID NO.3 is labeled with a fluorescent group of VIC, the 5 ' end of the fluorescent probe shown in SEQ ID NO.8 is labeled with a FAM fluorescent group, and the 3 ' ends of the two fluorescent probes are labeled with quenching groups of NFQ-MGB.
In one embodiment of the present invention, the reagents for synthesizing the first strand of cDNA comprise: MgCl2Reverse transcriptase buffer, dNTPs, RNase inhibitor, oligo (dT)15, reverse transcriptase and deionized water containing no RNase.
In an embodiment of the present invention, the fluorescent quantitative PCR pre-mix solution includes: mg (magnesium)2+dNTPs, deoxyuridine triphosphate dUTP, DNA polymerase, UNG enzyme and deionized water containing no ribonuclease.
In particular, Mg2+Including magnesium chloride and/or magnesium sulfate.
Specifically, the DNA polymerase includes: taq polymerase, KOD FX polymerase, Pfu polymerase or Phusion polymerase.
In one embodiment of the invention, the final concentration of each primer in the primer group is 0.2-1.0 μ M; the final concentration of the fluorescent probe in the primer group is 0.05-1.0 mu M. The method avoids that the final concentration of the upstream primer and the downstream primer and the fluorescent probe is too low to be beneficial to the amplification of the nucleic acid fragment, and also can avoid that the detection cost is too high due to too high final concentration.
For the final concentration of each primer, 0.2-1.0. mu.M refers to any value in the range of 0.2. mu.M to 1.0. mu.M, such as 0.2. mu.M, 0.4. mu.M, 0.6. mu.M, 0.8. mu.M, and 1. mu.M.
For the final concentration of the fluorescent probe, 0.05-1.0. mu.M means any value in the range of 0.05. mu.M to 1.0. mu.M, for example, 0.05. mu.M, 0.1. mu.M, 0.2. mu.M, 0.4. mu.M, 0.6. mu.M, 0.8. mu.M and 1. mu.M.
In one embodiment of the invention, the positive control sample comprises a plasmid carrying the detection fragment of the PML-RAR α fusion gene of type L, type V and type S to mimic a sample carrying a mutation of the PML-RAR α fusion gene.
Based on the primer group, the invention also provides a detection method of the PML-RAR alpha fusion gene, which comprises the following steps:
designing a primer set according to any one of the above, or removing the primer set in the kit according to any one of the above;
extracting RNA from a sample to be detected;
synthesizing cDNA by using the extracted RNA as a template, and using the cDNA as an amplification template;
preparing a fluorescent quantitative PCR reaction system containing the primer group and the amplification template;
carrying out fluorescent quantitative PCR amplification reaction on the fluorescent quantitative PCR reaction system to obtain an amplification curve map of a PCR product;
and determining the genotype of the PML-RAR alpha fusion gene mutation of the sample to be detected according to the amplification curve map.
Specifically, the fluorescent quantitative PCR reaction system comprises a primer group, an amplification template and Mg2+dNTPs, deoxyuridine triphosphate dUTP, DNA polymerase, UNG enzyme and deionized water containing no ribonuclease.
Specifically, when cDNA is synthesized using the extracted RNA, it can be synthesized by the first strand cDNA synthesis reagent and the extracted RNA in the above-described kit.
Specifically, the sample to be tested includes peripheral blood, bone marrow fluid or bone marrow blood of the body.
Specifically, the copy number of the ABL reference gene is an important index for experimental quality control, so that the Ct value of the ABL reference gene is less than or equal to 31, and the situation that the test result of a sample to be tested cannot be truly reflected due to the fact that the concentration of the ABL reference gene is too low is avoided.
When the gene typing of the PML-RAR alpha fusion gene of the sample to be detected shows a positive result, the fluorescent quantitative curve in the amplification curve map of the PCR product shows a smooth S shape, and the Ct value of the curve is less than or equal to 34.
When the PML-RAR alpha fusion gene of the sample to be detected has a negative genotyping result, a fluorescence quantitative curve in an amplification curve map of a PCR product is not drawn; or curve Ct value > 34.
In an embodiment of the present invention, the reaction conditions of the fluorescent quantitative PCR amplification reaction are: reacting for 60-300 s at 45-55 ℃; pre-denaturation at 94-96 ℃ for 120-900 s; denaturation is carried out for 5-20 s at the temperature of 93-97 ℃; annealing at the temperature of 58-62 ℃ for 20-180 s; repeating the denaturation and annealing for 35-50 times.
For the temperature of the enzyme used in the degradation instrument before the fluorescent quantitative PCR reaction, 45 to 55 ℃ means any value in the range of 45 ℃ to 55 ℃, for example, 45 ℃, 48 ℃, 50 ℃, 52 ℃, 54 ℃ and 55 ℃.
For the time for degrading the enzyme in the instrument before the fluorescent quantitative PCR reaction, 60-300 s means any value in the range of 60s to 300s, such as 60s, 100s, 150s, 200s, 250s and 300 s.
For the denaturation temperature, 94-96 ℃ means any value in the range of 94 ℃ to 96 ℃, such as 94 ℃, 95 ℃ and 96 ℃.
For the time of denaturation, 120-900 s refers to any value within the range of 120 s-900 s, such as 120s, 180s, 200s, 250s, 300s, 350s, 400s, 450s, 500s, 550s, 600s, 650s, 700s, 750s, 800s, 850s, and 900 s.
For the denaturation temperature, 93-97 ℃ means any value within the range of 93 ℃ to 97 ℃, for example, 93 ℃, 94 ℃, 95 ℃, 96 ℃ and 97 ℃.
For the time of denaturation, 5-20 s means any value in the range of 5s to 20s, for example, 5s, 8s, 10s, 12s, 14s, 16s, 18s, and 20 s.
For the annealing temperature, 58-62 ℃ means any value in the range of 58 ℃ to 62 ℃, for example, 58 ℃, 59 ℃, 60 ℃, 61 ℃ and 62 ℃.
For the annealing time, 20 to 180s means any value in the range of 20s to 180s, for example, 20s, 30s, 50s, 80s, 100s, 120s, 140s, 160s, and 180 s.
For the number of repetition of denaturation and annealing, 35 to 50 times means any value in the range of 35 to 50 times, for example, 35 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times and 50 times.
It should be noted that the invention is applicable to any common fluorescence quantitative PCR instrument, specifically including ABI fluorescence quantitative PCR instrument (ABI7300, ABI7500, ABI ViiA7, ABI Stepo plus (Stepo)); roche (Roche) fluorescent quantitative PCR instrument LightCycler 480; bio-rad (Berle) fluorescent quantitative PCR instruments CFX96TM and the like. Different fluorescent probes can be selected according to different PCR instruments for PCR amplification.
The detection method has the following advantages and effects:
(1) the fusion gene is detected by using a Taqman-MGB specific probe, so that the specificity is strong and the result accuracy is high.
(2) The whole detection process is carried out in a PCR tube, cover opening analysis is not needed, and aerosol pollution is not easily caused; electrophoresis analysis is not needed, and toxic and harmful reagents are not contacted.
(3) The whole detection process from sample receiving, RNA extraction, cDNA synthesis to PCR amplification can be completed within 5 hours, a large amount of samples can be detected simultaneously, the detection efficiency is improved, and the time cost is saved.
The detection method can quickly, accurately and efficiently detect whether PML-RAR alpha fusion gene mutation occurs or not and judge the type of gene fusion.
The method for detecting the PML-RAR α fusion gene is a non-diagnostic method.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a map of ABL reference gene in a sample showing a negative result of PML-RAR α fusion gene according to an embodiment of the present invention;
FIG. 2 is a map of bcr1 type detection well of PML-RAR alpha fusion gene negative sample provided by an embodiment of the present invention;
FIG. 3 is a map of bcr2 type detection well of PML-RAR alpha fusion gene negative sample provided by an embodiment of the present invention;
FIG. 4 is a map of bcr3 type detection well of PML-RAR alpha fusion gene negative sample provided by an embodiment of the present invention;
FIG. 5 is a map of ABL reference gene in a positive sample of PML-RAR α fusion gene according to an embodiment of the present invention;
FIG. 6 is a map of bcr1 type detection well of PML-RAR alpha fusion gene positive sample provided by an embodiment of the present invention;
FIG. 7 is a map of bcr2 type detection well of PML-RAR alpha fusion gene positive sample provided by an embodiment of the present invention;
FIG. 8 is a map of bcr3 type detection well of PML-RAR α fusion gene positive specimen provided in one embodiment of the present invention.
Detailed Description
Specifically, the reagents used in the implementation of the invention are all commercial products, and the databases used in the implementation of the invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.
Example 1: design and Synthesis of primer set
Step 1.1: specific primers and specific fluorescent probes are respectively designed according to base sequences on exons of ABL reference gene, PML and RAR alpha. Wherein, the specific primers are designed at two ends of the fusion site of the fusion gene through Primer Premier 5.0, and the most suitable primers are selected through software analysis; the specific fluorescent probe is positioned in a region between a pair of primers, wherein the Tm value is 60-70 ℃, and is usually 5-10 ℃ higher than that of the primers. The 5 ' end of a fluorescent probe referred by an ABL reference gene is marked by a VIC fluorescent reporter group, the 5 ' end of the probe for detecting bcr1, bcr2 and bcr3 fusion genes is marked by an FAM fluorescent reporter group, the 3 ' end of the probe is marked by a non-fluorescent quenching group (NQF), and meanwhile, the probe is also connected with an MGB modifying group.
In addition, the fluorescent reporter group can be replaced according to actual conditions in actual operation.
The primers and fluorescent probes of this example are shown in Table 1 below:
TABLE 1
| Sequence name | Primer sequence 5 '-3' |
| SEQ ID NO.1 | TGGAGATAACACTCTAAGCATAACTAAAGGT |
| SEQ ID NO.2 | GATGTAGTTGCTTGGGACCCA |
| SEQ ID NO.3 | VIC-CCATTTTTGGTTTGGGCTTCACACCATT-NQF-MGB |
| SEQ ID NO.4 | TCTTCCTGCCCAACAGCAA |
| SEQ ID NO.5 | CCCCAGGAAGGTCATCAAGA |
| SEQ ID NO.6 | CCGATGGCTTCGACGAGTT |
| SEQ ID NO.7 | GCTTGTAGATGCGGGGTAGAG |
| SEQ ID NO.8 | FAM-AGTGCCCAGCCCTCCCTCGC-NQF-MGB |
Wherein, the 5 'end of the fluorescent probe shown in SEQ ID NO.3 is marked with a fluorescent group VIC, and the 3' end is marked with a quenching group NQF-MGB; the fluorescent probe shown in SEQ ID NO.8 is labeled with fluorescent group FAM at the 5 'terminal area, and is sequentially labeled with quenching group NQF and modifying group MGB at the 3' terminal area.
Step 1.2: and (3) synthesizing the primer group designed in the step 1.1.
Example 2: extraction of RNA from test samples
Step 2.1: RNA is extracted from a sample to be tested by using an RNA extraction Kit PAXgene Blood RNA Kit, QIAGEN (762174), the concentration and the purity of the RNA are determined by adopting NP80-touch (IMPLEN, Germany), and the extracted RNA is stored when the test result is within a preset range.
Example 3: preparing a fluorescent quantitative PCR reaction system by using the primer group synthesized in the step 1.2 and the RNA stored in the step 2.1
Step 3.1: using the RNA stored in step 2.1 as template and MgCl2cDNA is synthesized by reverse transcriptase buffer, dNTPs, RNase inhibitor, oligo (dT)15, reverse transcriptase and deionized water without RNase.
Step 3.2: formulated to contain MgCl2dNTPs, dUTP, DNA polymerase, UNG enzyme, deionized water containing no ribonuclease, cDNA obtained in step 3.1, and the primer set synthesized in step 1.2 to obtain a fluorescent quantitative PCR reaction system.
Specifically, the PCR reaction system of the ABL reference gene is shown in table 2 below.
TABLE 2
| Reagent composition | Volume of |
| 2×TaqMan PCR Mix | 9μl |
| ABL-F(10uM) | 0.8μl |
| ABL-R(10uM) | 0.8μl |
| ABL-Probe(10uM) | 0.8μl |
| cDNA | 1μl |
| Ultrapure water | 7.6μl |
Specifically, the PCR reaction systems for bcr1, bcr2, and bcr3 fusion genes are shown in Table 3 below.
TABLE 3
| Reagent composition | Volume of |
| 2×TaqMan PCR Mix | 9μl |
| PML-F*(10uM) | 0.8μl |
| RARA-R(10uM) | 0.8μl |
| RARA-Probe(10uM) | 0.8μl |
| cDNA | 1μl |
| Ultrapure water | 7.6μl |
Wherein, except that the upstream primer is different, the other reverse primers and the fluorescent probe are consistent with each other for the L type, the V type and the S type of the PML-RAR alpha fusion gene. Specifically, the upstream primer of the L type of the PML-RAR alpha fusion gene is as follows: PML-Bcr 1-L-F; the upstream primer of V type of PML-RAR alpha fusion gene is PML-bcr 2-V-F; the upstream primer of the S-type of the PML-RAR alpha fusion gene is PML-Bcr 3-S-F.
It should be noted that, the proportional scaling up/down of the reaction system is within the protection scope of the embodiment of the present invention; the amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate proportion.
Step 3.2: the procedure of the PCR instrument was set according to the fluorescent quantitative PCR reaction shown in Table 4 below, and the multiplex PCR amplification reaction was performed on the fluorescent quantitative PCR reaction system prepared in step 3.2 to obtain an amplification curve map of the PCR product.
TABLE 4
Where, indicated in table 4 denotes the positions where the fluorescence signals were received.
Specifically, the embodiment adopts the ABI7500 real-time fluorescence quantitative PCR instrument for amplification, but the invention is not limited to the ABI7500, and is also suitable for fluorescence quantitative PCR instruments of other models, and specifically, different fluorescence probes can be selected for detection according to different PCR instruments.
Example 4: judgment of detection result
Based on the starting peak and Ct values of the ABL reference gene, bcrl type, bcr2 type and bcr3 type in the amplification curve map obtained in example 3.2, the negative and positive and fusion gene typing were determined as follows:
wherein the Ct value of the sample ABL reference gene is less than or equal to 31.
If the gene typing of the PML-RAR alpha fusion gene of the sample to be detected shows a positive result, the fluorescent quantitative curve in the amplification curve map shows a smooth S shape, and the Ct value of the curve is less than or equal to 34.
If the gene typing of the PML-RAR alpha fusion gene of the sample to be detected is a negative result, the fluorescence quantitative curve in the amplification curve map is not drawn, or the Ct value of the curve is more than 34.
FIG. 1 to FIG. 8 show fluorescence PCR amplification curve maps of PML-RAR α fusion gene negative results of a test sample; FIGS. 5 to 8 show the fluorescence PCR amplification curve maps of the PML-RAR α fusion gene positive results of another test sample. The abscissa of fig. 1 to 8 is Cycle, and the ordinate is Δ Rn.
The missing figures in fig. 1 to 8 do not affect the technical content of the present solution.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.
Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (10)
1. A primer set for detecting a PML-RAR alpha fusion gene, comprising:
the primer pair is used for amplifying the ABL reference gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.1, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.2, and the sequence of a corresponding fluorescent probe is shown as SEQ ID NO. 3;
the primer pair for amplifying the PML-RAR alpha L type fusion gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.4, the nucleotide sequence of a downstream primer is shown as SEQ ID No.7, and the sequence of a corresponding fluorescent probe is shown as SEQ ID No. 8;
the primer pair for amplifying the PML-RAR alpha V type fusion gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.5, the nucleotide sequence of a downstream primer is shown as SEQ ID No.7, and the sequence of a corresponding fluorescent probe is shown as SEQ ID No. 8;
the primer pair for amplifying the PML-RAR alpha S type fusion gene is characterized in that the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.6, the nucleotide sequence of a downstream primer is shown as SEQ ID No.7, and the sequence of a corresponding fluorescent probe is shown as SEQ ID No. 8.
2. A kit for detecting a PML-RAR α fusion gene, comprising: the primer set of claim 1, a first strand cDNA synthesis reagent, a quantitative fluorescent PCR premix for PCR Polymerase Chain Reaction (PCR) assay, a negative control sample, and a positive control sample, wherein the negative control sample is a sample without PML-RAR α fusion gene mutation, and the positive control sample is a sample with PML-RAR α fusion gene mutation.
3. The kit according to claim 2,
the fluorescent probe shown as SEQ ID NO.3 and the fluorescent probe shown as SEQ ID NO.8 in the primer group are marked with fluorescent groups at the 5 'terminal area and are marked with quenching groups at the 3' terminal area;
wherein the fluorescent group includes any one of FAM, TET, VIC, ROX, Texas Red-X, Cy3 and Cy 5.
4. The kit according to claim 3,
the quencher group comprises: TAMRA, BHQ, DABCYL, and NFQ-MGB.
5. The kit according to claim 2,
the first strand cDNA synthesis reagent comprises: MgC12Reverse transcriptase buffer, dNTPs, RNase inhibitor, oligo (dT)15, reverse transcriptase and deionized water containing no RNase.
6. The kit according to claim 2,
the fluorescent quantitative PCR premix solution comprises: mg (magnesium)2+dNTPs, deoxyuridine triphosphate dUTP, DNA polymerase, UNG enzyme and deionized water containing no ribonuclease.
7. The kit according to claim 2,
ct value of ABL reference gene in the primer group is less than or equal to 31.
8. The kit according to any one of claims 2 to 7,
the final concentration of each primer in the primer group is 0.2-1.0 mu M;
the final concentration of the fluorescent probes in the primer group is 0.05-1.0 mu M;
and/or the presence of a gas in the gas,
the positive control sample comprises plasmids with detection fragments of PML-RAR alpha fusion genes of L type, V type and S type.
The detection method of the PML-RAR alpha fusion gene is characterized by comprising the following steps:
designing the primer set of claim 1, or removing the primer set in the kit of any one of claims 2 to 8;
extracting RNA from a sample to be detected;
synthesizing cDNA by using the extracted RNA as a template, and using the cDNA as an amplification template;
preparing a fluorescent quantitative PCR reaction system containing the primer group and the amplification template;
carrying out fluorescent quantitative PCR amplification reaction on the fluorescent quantitative PCR reaction system to obtain an amplification curve map of a PCR product;
and determining the genotype of the PML-RAR alpha fusion gene mutation of the sample to be detected according to the amplification curve map.
10. The method of claim 9,
the reaction conditions of the fluorescent quantitative PCR amplification reaction are as follows: reacting for 60-300 s at 45-55 ℃; pre-denaturation at 94-96 ℃ for 120-900 s; denaturation is carried out for 5-20 s at the temperature of 93-97 ℃; annealing at the temperature of 58-62 ℃ for 20-180 s; repeating the denaturation and annealing for 35-50 times.
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